Covalent Proteomimetic Inhibitor of the Bacterial FtsQB Divisome Complex

The use of antibiotics is threatened by the emergence and spread of multidrug-resistant strains of bacteria. Thus, there is a need to develop antibiotics that address new targets. In this respect, the bacterial divisome, a multi-protein complex central to cell division, represents a potentially attractive target. Of particular interest is the FtsQB subcomplex that plays a decisive role in divisome assembly and peptidoglycan biogenesis in E. coli. Here, we report the structure-based design of a macrocyclic covalent inhibitor derived from a periplasmic region of FtsB that mediates its binding to FtsQ. The bioactive conformation of this motif was stabilized by a customized cross-link resulting in a tertiary structure mimetic with increased affinity for FtsQ. To increase activity, a covalent handle was incorporated, providing an inhibitor that impedes the interaction between FtsQ and FtsB irreversibly. The covalent inhibitor reduced the growth of an outer membrane-permeable E. coli strain, concurrent with the expected loss of FtsB localization, and also affected the infection of zebrafish larvae by a clinical E. coli strain. This first-in-class inhibitor of a divisome protein–protein interaction highlights the potential of proteomimetic molecules as inhibitors of challenging targets. In particular, the covalent mode-of-action can serve as an inspiration for future antibiotics that target protein–protein interactions.


Olefin crosslink
For macrocyclization of olefinic non-natural amino acids, ring closing metathesis was performed. 4 Fmocprotected non-natural olefinic amino acids (Okeanos Tech, Beijing, China) were incorporated in peptide synthesis and treated as natural amino acids. After synthesis, the resin with immobilized peptide was washed and swollen in dichloroethane (DCE) for 15 min. A solution of 4 mg·mL -1 benzylidene-bis(tricyclohexylphosphine)dichlororuthenium (Grubbs Catalyst TM 1st generation) in DCE was added to the resin and reacted at room temperature (RT) for 1.5 h. This procedure was repeated until a sufficient quantity of crosslinked peptide was observed in analytical LC/MS. After metathesis, the resin was washed with DCE, dichloromethane (DCM) and DMF three times, respectively.

Protein expression and purification
Plasmids were obtained from Luirink et al. as described in previous publications. 5

Transformation and expression
Escherichia coli BL21 DE3 were freshly transformed with pET16b FtsQ(50˗276), a His6 fusion construct of the periplasmic domain of FtsQ in a modified pET16b vector. After heat shock transformation, bacteria were grown on an agar plate with 100 μg·mL -1 ampicillin as selection marker at 37 °C overnight. A single colony was used to inoculate 100 mL lysogeny broth (LB) medium (1 g tryptone, 0.5 g yeast extract, 1 g NaCl, add 100 mL ddH2O, pH 7.4) with 100 µg·mL -1 ampicillin. Cells were grown at 37 °C and 200 rpm overnight. 50 mL of this overnight pre-culture were used to inoculate 1 L LB medium (100 μg·mL -1 ampicillin) and incubated at 37 °C and 200 rpm.
After the culture reached an optical density (OD600) of 0.8 (λ = 600 nm), expression was induced with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) and the cells were grown until an OD600 of 1.5 was reached. After harvesting the cells by centrifugation at 4000 rcf and 4 °C for 15 min, the cell pellet was washed in 6 mL phosphate-buffered saline solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). The suspension was centrifuged, the supernatant was discarded and the washed pellet was shock frozen in liquid N2 and stored at -80 °C until further use. For protein purification, the pellet was resuspended in buffer I (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 4 % glycerol and 10 mM imidazole) to a total volume of 100 mL, before DNase, lysozyme and 100 µM PMSF were added. The mixture was incubated on ice for 5 min. The cells were then disrupted using the Microfluidizer 1109 (15000 PSI). After four cycles of homogenization, the cell debris was removed by centrifugation at 70.000 rcf at 10 °C for 45 min.

Isolation and purification
To isolate FtsQ(50˗276), the soluble fraction was applied to a 5 mL HiTrap TALON crude prepacked column (Cytivia) and purification performed on an AKTA FPLC system (Cytivia). After a wash with 2 column volumes (CV) buffer I the His6-fusion protein was eluted with high imidazole buffer II (50 mM sodium phosphate pH 7.5, 500 mM NaCl, 4 % glycerol, 400 mM imidazole) using a stepwise gradient (Gradient: 1 CV 5% buffer II, 5 CV 24.3% buffer II, 2 CV 100% buffer II; 2 mL fractions). In a final step, the buffer was exchanged and the protein concentrated in buffer III (10 mM HEPES, pH 7.4, 150 mM NaCl and 4 % glycerol) using Amicon Ultra Centrifugal Filters (10 kDa cut off). After concentration FtsQ was frozen in liquid nitrogen and stored at -80°C in 10 mM HEPES, pH 7.4, 100 mM NaCl, 4 % glycerol. Fresh aliquots were used in every experiment. 2

Fluorescence polarization assay for affinity measurements
To determine the affinity of the peptides their FITC-labelled versions were dissolved in assay buffer (

Molecular Dynamics (MD) parameterization and simulations
A structure of the FtsB-bound 24f peptide was designed using Avogadro software 6 utilizing the crystal structure of the FtsQ-FtsB complex as a template (PDB: 6h9o). The Cβ atom of R72 was connected to the Cβ of E82 by the cross-linker (residue name STP) containing a total of 10 additional carbon atoms, including a trans double bound between the central 5 th and 6 th carbon atoms. The geometry of the staple was optimized, avoiding clashes either internally or with the bound FtsB molecule. The arginine and glutamate residues involved in bonding to the staple were re-defined as modified alanine residues (name AST) for parameterization.
GAFF-based (general AMBER force field) parameters were obtained to describe the staple structure. Initially, partial charges were derived using antechamber (Amber tools 7 ) using the AM1-BCC charge model, calculated for a methyl-capped structure of the staple. Complete parameters for the 10-carbon staple were generated using the prepgen application, with additional parameters added manually to describe the bonding between the staple and connecting side chains. Amber parameters for the non-canonical norleucine residue within the peptide were available and applied from the Forcefield_NCAA 8 library. All relevant parameters, input files, and structural snapshots are included in the following Github depository (https://github.com/georgehutch/FtsQ_staple_MD).
Multiple independent MD simulations of FtsB in complex with both wild-type FtsQ and stapled 24f were calculated, including three 100 ns and one 400 ns simulations of the FtsB/24f complex to ensure thorough sampling of potential binding poses. Protonation of the complex was determined using the H++ server 9 and input files were generated using tleap including solvation and neutralization in TIP3P water box, followed by system minimization, heating and NPT equilibration. Production MD simulations were executed with the Amber20 pmemd.cuda application using the Bazis HPC cluster (VU Amsterdam) and trajectories analyzed using cpptraj.

Protein modification assay
To determine the reactivity of the different modifiers FtsQ(50˗276) was diluted to a concentration of 100 µM in 20 mM NH4HCO3 buffer (pH 7.6, 150 mM NaCl). The individual peptides were diluted to a concentration of 250 μM in the same buffer. Equal volumes of both solutions were mixed and incubated at 37 °C for the given time period (1 h or 3 h). The reaction was quenched using 5x SDS sample buffer (pH 6.8, 1.875 g TRIS-Cl, 25 mL glycerol, 5 g SDS, 12.5 mL β-mercaptoethanol, 0.5 % bromphenol blue and add 50 mL ddH2O). Samples were heated to 96 °C for 2 min and subsequently analysed by 17 % tris/tricine PAGE (15 min at 90 V and then 3:45 h at 150 V). PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (Thermo Scientific™) was used as a ladder.

LC/MS-analysis of modified FtsQ(50˗276)
To determine the reactivity of 17fα, FtsQ(50˗276) was diluted to a concentration of 100 μM in 20 mM NH4HCO3 buffer (pH 7.6, 150 mM NaCl). The peptide 17fα was diluted to a concentration of 250 μM in the same buffer.
Equal volumes of both solutions were mixed and incubated at 37 °C for the given time period ( performed by adding 50 μL of the diluted peptide to the top row, mixing it and taking 50 μL to the next row. At each step the solution was resuspended five times. This was repeated until the last row which was skipped in order to obtain a reference measurement. After 2-3 h, the E.coli lptD4213 (imp) pre-culture was diluted to an OD600 of 0.00005. Then, 50 μL of diluted culture were added to each well and the plate sealed using a lid and parafilm. The OD600 was measured every 15 min over a period of more than 20 h (continuously shaking, 37°C) using a BioTek Synergy H1 plate reader. The obtained data were analyzed and relative growth curves plotted with GraphPad Prism 8. The assays were performed as technical triplicates. A single overnight pre-culture was used to prepare the diluted MOPS culture used in the growth assay. The peptides of interest were diluted to the desired concentrations separately for each triplicate measurement. ^

Microscopy and morphology assay
Morphology assays were executed to assess the physiological change of E.coli cells upon exposure to different peptides. All steps in this protocol were performed under sterile conditions. For each assay, 20 mL of fresh MOPS medium was prepared (40 mM MOPS, 4 mM tricine, 0.01 mM FeSO -4 , 9.5 mM NH4Cl, 0.276 mM K2SO4, 0.5 μM CaCl2, 50 mM NaCl, traces of (NH4)6Mo7O24, H3BO3, CoCl2, CuSO4, MnCl2 and ZnSO4, 1.32 mM dibasic anhydrous KH2PO4, 0.20% glucose, 1.5 mM KOH, 0.199 mM adenine, cytosine, uracil and guanine and 0.01 mM to 10 mM amino acids). A preculture of E.coli lptD4213 (imp) was prepared in 5 mL MOPS medium with streptomycin (30 μg/mL), which was grown overnight at 200 rpm and 37°C. The next day, the overnight culture was diluted a thousandfold and grown up to an OD600 of about 0.5 at 200 rpm and 37°C. After 2-3h, the culture was diluted to an OD600 of 0.1. 10 mM peptide stocks in DMSO were subsequently diluted to 200 μM in MOPS medium. For measuring two time points, a total volume of 110 μL peptide dilution was prepared, for 3 timepoints this was 160 μL. For each time point, 50 μL of diluted peptide was added into an Eppendorf tube, to which 50 μL diluted culture was added (final peptide concentration: 100 μM, starting OD600 = 0.05). The tubes were incubated for either 1,3 or 6 h at 200 rpm and 37°C. Then the cells were fixed using 8.3 μL 37% formaldehyde solution in H2O and incubated for 5 min at 200 rpm and 37°C. Subsequently, the mixture was centrifuged at 500 rpm for 5 min, after which the supernatant was discarded. The pellets were redissolved in 7.5 μL PBS buffer and stored at -20°C. 2 Microscopy slides were prepared by melting 1% Nobel Agarose in MiliQ water in a microwave. 800 μL was dispersed over a well slide, a microscopy slide was put on top and air bubbles were removed. The agar was allowed to cool for 15 min, after which the microscopy slide was removed. The frozen samples were thawed and 3 μL of each sample was added to separate wells, after which a covering glass was put on top. Using an Olympus IX83 microscope with 100 times magnification ocular and phase contrast (halogen lamp wat approximately 7V), the morphology of the cells was investigated. Pictures were taken and subsequently analyzed with ImageJ and the ObjectJ Cell Counter (https://sils.fnwi.uva.nl/bcb/objectj/examples/CellCounter/cellcounter-md/cellcounter.html, accessed on February 2020).  FICindex ≤ 0.5 was considered synergistic, whereas FICindex ≥ 2 was considered antagonistic. An FICindex between 0.5 and 1 was considered to be an additive effect.

Supporting tables
Corresponding peptide sequences are listed in Supporting Table S1.